Method and apparatus for detection and measurement of particles with a wide dynamic range of measurement

ABSTRACT

Light from a light source is directed at a flow path of particles of a flow cytometer. The directed light results in a light pattern having a plurality of lobes. A first signal is detected exceeding a first threshold. A second signal exceeding a second threshold is detected, wherein the second threshold is greater than the first threshold. Based on detecting the second trigger after detecting the first trigger, is determined that the first and second signals were created by a relatively large particle.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. Non-Provisional applicationSer. No. 14/332,347, filed Jul. 15, 2014, and also claims the benefit ofU.S. Provisional Application No. 61/957,887, filed Jul. 15, 2013, whichis incorporated by reference.

BACKGROUND OF THE INVENTION

In a flow cytometer, a focused light source such as a laser beam or LEDis arranged to shine on particles flowing within a fluid stream. Theparticles scatter the light and also cause fluorescent light of adifferent wavelength than the incident beam to be emitted.

The focused spot however is usually not a bright point that decays awaymonotonically in all directions. Rather, the beam from the light sourcesuch as a laser can have “lobes.” The far field of the beam is typicallythe Fourier transform of the laser aperture. Thus, for example a squareaperture will give a far field which is a 2 dimensional Sync function asshown in FIG. 1. The beam shaping optical elements may also have smallreflections from various surfaces as shown in FIG. 3, coatings canreduce the reflections but typically will not completely eliminate them.Both of these effects have the net effect of causing the laser beam inthe flow channel 17 to have a main peak 3 flanked on one or both sidesby many smaller peaks 4 as shown in FIG. 3 and FIG. 2.

While the power in a laser minor side lobe 4 is typically 1% or less ofthe main lobe 3, a difficulty arises when very large particles 20 andvery small particles 21 as shown in FIG. 2 are mixed. A large particlepassing over a minor lobe of the laser will cause an amount of signalthat is comparable to a small particle that is passing the main lobe ofthe laser. A large particle passing over a minor lobe of the laser andthen subsequently passing over the main lobe of the laser canincorrectly be counted as a small particle followed by a large particle.

BRIEF SUMMARY OF THE INVENTION

This invention relates to the field of cytometry. It is sometimesnecessary to detect particles of widely varying size, typically from 50nm to 50 μm a dynamic range of 1000×. According to many embodiments,particle parameter measurement which may be recalculated instantaneouslyor at a later time, dependent on the determination as to if the particlewas a large or small particle.

Many embodiments relate to an analog or digital deconvolution filterthat can operate on a light intensity data pattern received from lightemissions in a flow channel, so as to mitigate the effect of the laserlobes or other stray light in the flow channel caused by opticalreflections or imperfections.

Many embodiments relate to a particle detection trigger mechanism thatmay be retriggered or restarted depending on measured values of datareaching fulfilling certain conditions requiring restart.

Many embodiments relate to a detection system in which laser light isdirected at a flow path of particles of a flow cytometer. The laserlight results in a light pattern having a plurality of lobes. A firstsignal is detected exceeding a first threshold. A second signalexceeding a second threshold is detected, wherein the second thresholdis greater than the first threshold. Based on detecting the secondtrigger after a particular time point after detecting the first trigger,is determined that the first and second signals were created by a singlerelatively large particle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing of a two dimensional Sync function.

FIG. 2 is a drawing of the light pattern intersecting the flow channel.

FIG. 3 is a drawing of secondary lobes being created by reflections fromoptical elements.

FIG. 4 is a drawing of two laser beams of different colors intersectinga flow channel, according to many embodiments.

FIG. 5 is a system diagram of a flow cytometer system, according to manyembodiments.

FIGS. 6A and 6B are flow charts of methods for detecting a particle,according to many embodiments.

FIG. 7 is a system diagram of a computing system, according to manyembodiments.

DETAILED DESCRIPTION OF THE INVENTION

In most cases, flow cytometers operate by flowing particles inside asmall channel 17 formed in a middle of typically a quartz cuvette 12 asshown in FIG. 4. In most cases the surfaces of quartz surfaces formingthe inside of the cuvette and the outside of the cuvette are highlypolished and flat such that a laser beam may be shone through andfocused at one point in the cross section of the channel with opticalprecision. The particles flowing past the light source, such as a laserbeam then cause scattered light emissions of the same wavelength of thelaser, or fluorescent light emissions of a higher wavelength than of thelaser. These light pulses are detected by one or more light detectors 11such as Photomultipliers or Photodiodes with filters that only pass thewavelength(s) of interest. The pulsations last an amount of timedetermined by the flow rate in the channel and the size of the particleand the width of the laser spot. The time domain waveform of thescattered or fluorescent light being the convolution of the spatialdistribution of the scattering or fluorescent emission of the particlewith the spatial distribution of the laser light pattern, theconvolution being done at a rate determined by the flow rate.

For simplistic analysis let us first consider a “Particle A” having animpulse as the spatial distribution of 0,1,0. It can be seen that if weconvolve this particle pattern with a “Laser A” having an impulsespatial distribution of 0,1000,0 the resulting signal is 0,1000,0.Similarly if we convolve a “Laser B” with spatial distribution 0,1,0with a “Particle B” of impulse spatial distribution 0,1000,0, theresultant signal is 0,1000,0. In this simple example, both cases causean identical and indistinguishable signature of emitted light. In a morerealistic case a particle that scatters more may be larger and have awider spatial signature such as 0,0,1,10,100,1000,100,10,1,0,0.

A problem occurs when the laser spot spatial intensity variation in thecuvette is not an impulse, but instead is non-monotonically decayingundulating waveform as shown in FIG. 1 and FIG. 2. Such undulations arequite natural. It is well known that the far field of a laser or ingeneral any source of electromagnetic radiation, is the Fouriertransform of the spatial distribution of the electromagnetic energy atits emission aperture. The mathematical summation of the phase shiftedenergy received at a distant point (far field) from each emission pointbeing mathematically equivalent to a Fourier transform. Thus, forexample a uniform square emission aperture will yield a Sync function inX and in Y. A round aperture will yield a Bessel function. Both of theabove functions have “Minor Lobes” in the far field with zeros inbetween the lobes.

Minor laser lobes or their equivalent can also be created by near normalincidence of a laser beam 5 to an optical element 2 as shown in FIG. 3,due to reflected beams 4 in addition to the main beam 3. Imperfectionsin the light path can also cause minor lobes. Anti-reflective coatingscan reduce the reflection but not eliminate it, especially in the caseof simultaneous presence of multiple laser emission sources of differentwavelengths, where the coating must necessarily be a compromise.

Typically as shown in FIG. 4 several laser beams of different colors 15,16 are arranged along the flow path and optical element(s) 10 are usedto capture any emitted light 14 as the particle flows past each of thelaser beams 15 and 16. Thus, the light from any laser and its lobes willcause an electrical signal output from a photo-detector as long as it isof a wavelength that is allowed to reach the photo-detector. Differentoptical architectures can minimize this effect by using designedincluding but not limited to pinholes or to limit the amount of lightcollected away from the center of the laser but these designs do notblock all of the light emitted by the laser light outside of a specifiedregion.

Considering a flow cytometry system with a single laser and nearbyside-lobes: First consider a “Particle A” with a spatial pattern that isan impulse of 0,1,0 convolved with a “Laser C” of spatial pattern0,1,0,1000,0,1,0 (the ones on either side of the 1000 being the sidelobes), the convolution yields 0,1,0,1000,0,1,0. Now consider a“Particle B” with a spatial pattern of 0,1000,0, convolved with the samelaser pattern for “Laser C” the convolution yields0,1000,0,1000000,0,1000,0. With a triggering threshold of 1000, one candetect the particle with the spatial pattern of 0,1,0 correctly,however, one will falsely detect the response to the lobe only for theparticle with a spatial pattern of 0,1000,0, further, these particlesmay be “aborted” completely with a low trigger level because they may beclassified as “doublets”—i.e. the presence of two particles in the laserbeam simultaneously. In certain applications, it can be necessary to beable to distinguish between small particle entering the main lobe and a“larger” particle entering a side lobe. As used herein, the term“larger” not necessarily meaning physically larger but instead could bea particle of the same size but with more fluorescence or scattering.

Certainly a physically larger “Particle C” can have a spatial patternlike 0,0,1,10,100,1000,100,10,1,0,0 which indicates a relatively largephysical size. Convolution of this particle with a “Laser C” withpattern 0,1,0,1000,0,1,0, yields1,10,1100,11000,100101,1000000,100101,11000,1100,10,1 in which case theoutput signal is monotonically increasing and decreasing with a singlepeak even though the laser lobes are non-monotonic.

Depending on the spatial distribution of the laser and the spatialdistribution of the particle, and the relative sizes of the light lobescompared to the particle size, it is possible for a single particle tocreate several blips that may be mistakenly interpreted as severaldifferent particles.

With knowledge of the laser lobe structure within the cuvette it ispossible to mitigate the effect of the lobes. The essence of this methodis “deconvolution” of the spatial distribution of the scattering orfluorescent emission of the particle with the spatial distribution ofthe laser light pattern.

In an analog cytometer system 500 as shown at FIG. 5, (having a lasersystem 502, flow path 504, photodiode/amplifier circuit 506, integrationlogic circuits 510 with analog integrators and the like). An exemplaryflow cytometer system is disclosed at U.S. Pat. No. 8,570,500, which isincorporated by reference. By way of example but not limited to: Havingknowledge of the relative amplitudes and positions of the main and sidelobes it is possible to create a deconvolution logic circuit 508, by wayof example having two analog comparators within one set to the actualtrigger threshold desired for detection of all particles, and anotherset somewhat higher.

The logic 508 will classify any trigger event on the lower trigger, onlywhen followed by a trigger on the higher trigger at a time separated bythe distance of the laser lobes, to be a false first trigger of a largeparticle entering a side lobe, and requiring an immediate restart of theintegrators based on the higher trigger level, since the higher triggerindicates a large particle in the main lobe of the laser, and not a sidelobe. Comparatively, a smaller particle would not trigger on the hightrigger on the side lobe, because the energy provided by the side lobeis not high enough to trigger the lower trigger, but only trigger at themain lobe.

Typically reset of integration logic 510 for determination of peakheight and area is not required because the side lobe energy is so lowthat the main lobe can be integrated right on top of the side lobeintegration, which started based on the lower trigger just previously,with less than 1% error. However, calculations such as width, which isdetermined by integrating a constant current onto a capacitor for theduration of the pulse, can require restart of the integration.

Alternatively, for a digital cytometer system where the entire datastream is digitized it is possible to process captured data through aninverse deconvolution filter by way of a processor shown at FIG. 7. Anexemplary deconvolution filter is disclosed by Steven W. Smith, TheScientist & Engineer's Guide to Digital Signal Processing, 1st edition(1997), (e.g., Chapter 17), which is incorporated by reference in itsentirety. Here, spatial convolution is occurring between two spatialpatterns that yields a time domain signal. One of the spatial patternsis the light (laser) emission pattern. The other spatial pattern is theemitted energy pattern of the particle. Hence, L(x)*P(x)=>S(t) whereL(x) is the laser spatial pattern (spatial meaning in the dimensionx), * is the convolution integral, P(x) is the particle spatial pattern,and S(t) is the time domain signal received. Accordingly, knowing L(x)and S(t) the signal can be deconvoluted. This can be done prior toprocessing any parameters for the received pulse such as Peak, Area, LogArea, Log peak, Width, etc.

FIG. 6A shows a flow chart of a method 600 that can be implemented byway of an analog system per FIG. 5. At operation 602, laser light isemitted at a flow cytometer particle flow path resulting in a lightpattern of lobes. Generally, a main center lobe is created, which isflanked by side lobes, due to limitations (e.g., inherent laserlimitations, internal reflection in the cuvette caused by imperfections,chips or scratches, debris/dust/fuzz on the cuvette, inside, or outsidewall, and dust on the excitation optics) of the cytometer system

At operation 604, relatively small and large particles are flowedthrough a flow path. Relatively small particles can be, for example, assmall as 50 nm while relatively large particles can be as large as 50 μmparticles, which is a dynamic range of 1000×. A detection systemmonitors for energy signals (fluorescence and/or scatter) resultant fromparticles interfacing with the light pattern of lobes. Generally, thecenter lobe provides greater energy than the shown side lobes. Hence, arelatively small particle will trigger a given threshold only whenexcited by the center lobe, while a relatively large particle willtrigger the same threshold when passing through a side and center lobes,thus, causing potential measurement errors.

At operation 606, a first signal is detected that has an energy levelexceeding a first threshold. The first threshold can be set low enoughto detect a relatively small sized particle passing through the mainlobe.

At operation 608, a second signal is detected (successively after thefirst signal) that has an energy level exceeding a second threshold thatis higher than the first threshold. The second threshold can be set at alevel to detect a relatively large particle passing through the mainlobe. Hence, a small particle would not trigger the second threshold. Ifthe second threshold was not triggered by the second signal, theparticle would be processed as a small particle.

With both thresholds being triggered, it is still unknown whether thetriggers occurred from a single large particle passing through multiplelobes, or from a large particle passing through a side lobe directlyfollowing a small particle passing through a main lobe. Accordingly, atoperation 610, the detection system analyzes whether the second signalwas received at a predetermined time after the first signal according tothe time of particle flight between adjacent lobes. A large particlewould trigger at a side lobe and main lobe between an expected timedifferential, since the flow rate and distance between lobes are bothconstant. While different particles would more than likely be separatedat a distance that is not exactly coincident with the distance betweenlobes.

When it is determined that the first and second signals are resultantfrom a relatively large particle according to the time information, arequisite integration is performed to measure characteristics of thelarge particle at operation 612. When it is determined that the firstand second signals are resultant from a relatively large particledirectly following a relatively small particle, requisite integrationsare performed to measure characteristics of the both particles atoperation 614.

FIG. 6B shows a method 616 for detecting a particle using any of thesystems disclosed herein. At operation 618, laser light is emitted at aflow cytometer particle flow path resulting in a spatial light patternof lobes. Generally, a main center lobe is created, which is flanked byside lobes, due to limitations (e.g., inherent laser limitations,internal reflection in the cuvette caused by imperfections, chips orscratches, debris/dust/fuzz on the cuvette, inside, or outside wall, anddust on the excitation optics) of the cytometer system

At operation 620, relatively small and large particles are flowedthrough a flow path. Relatively small particles can be, for example, assmall as 50 nm while relatively large particles can be as large as 50 μmparticles, which is a dynamic range of 1000×. A detection systemmonitors for energy signals (fluorescence and/or scatter) resultant fromparticles interfacing with the light pattern of lobes.

At operation 622, a time domain signal having a spatial emission patternis detected from one or more particles interacting with the plurality oflobes. However, size of the one or more particles is not discernible inthe emission pattern due to convolution between the spatial lightpattern (plurality of lobes) of the laser and the spatial emissionpattern of the particle.

At operation 624, the spatial emission pattern is deconvoluted (e.g.,using a deconvolution filter as described above) from the time domainsignal, based on dimensions of the plurality of lobes, to discern thesize or spacing of the one or more particles.

FIG. 7 is a high level block diagram of a computing system 700 that maybe used to implement any of the entities or components described above,which may include one or more of the subsystems or components shown inFIG. 5. The subsystems shown in FIG. 5 are interconnected via a systembus 705. Additional subsystems such as a printer 710, keyboard 715,fixed disk 720, monitor 725, which is coupled to display adapter 730,and others are shown. Peripherals and input/output (I/O) devices, whichcouple to an I/O controller 735, can be connected to the computingapparatus 700 by any number of means known in the art, such as port 740(e.g., USB, serial, etc.) For example, port 740 or external interface745 can be used to connect the computing apparatus 700 to a wide orlocal area network, the Internet, a mouse input device, or any of thesubsystems shown at FIG. 5 for control thereof. The interconnection viathe system bus 705 allows the central processor 750 to communicate witheach subsystem and to control the execution of instructions from systemmemory 755 or the fixed disk 720, as well as the exchange of informationbetween subsystems. The system memory 755 and/or the fixed disk 720 mayembody a computer readable medium.

It should be understood that the present invention as described abovecan be implemented in the form of control logic using computer softwarein a modular or integrated manner. Based on the disclosure and teachingsprovided herein, a person of ordinary skill in the art can know andappreciate other ways and/or methods to implement the present inventionusing hardware and a combination of hardware and software.

Any of the software components, user interfaces, or methods described inthis application, may be implemented as software code to be executed bya processor using any suitable computer language such as, for example,Java, C++ or Perl using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructions,or commands on a computer readable medium, such as a random accessmemory (RAM), a read only memory (ROM), a magnetic medium such as ahard-drive or a floppy disk, or an optical medium such as a CD-ROM. Anysuch computer readable medium may reside on or within a singlecomputational apparatus, and may be present on or within differentcomputational apparatuses within a system or network.

What is claimed is:
 1. A method for particle detection comprising:directing a light source at a flow path of particles of a flowcytometer, wherein the particles comprise first particles and secondparticles, wherein the second particles are larger than the firstparticles, and wherein the directed light results in a light patternhaving a plurality of lobes in the flow path; detecting a first signalexceeding a first threshold, wherein the first signal is caused by theparticles flowing through a first lobe of the plurality of lobes of thelight pattern; detecting a second signal exceeding a second threshold,wherein the second signal is caused by the particles flowing through asecond lobe of the plurality of lobes of the light pattern, and whereinthe second threshold is greater than the first threshold; and based ondetecting the second signal and detecting the first signal at differenttimes, determining that the first and second signals were created by aparticle of the second particles.
 2. The method of claim 1, furthercomprising determining that the second signal was received at apredetermined time after the first signal according to particle travelspeed between adjacent lobes of the plurality of lobes.
 3. The method ofclaim 1, further comprising resetting one or more integrators configuredto process a particle of the first particles or to process a particle ofthe second particles.
 4. The method of claim 1, wherein the secondparticles are up to 1000 times larger than the first particles.
 5. Themethod of claim 1, wherein the first and second signals are detected byan analog logic system.
 6. The method of claim 1, wherein the firstthreshold is set according to an energy emission level of a particle ofthe first particles.
 7. The method of claim 6, wherein the secondthreshold is set according to an energy emission level of a particle ofthe second particles.
 8. A particle detection system comprising: acytometer system configured for directing light source at a flow path ofparticles, wherein the particles comprise first particles and secondparticles, wherein the second particles are larger than the firstparticles, and wherein the light results in a light pattern having aplurality of lobes in the flow path; and a detection system configuredfor: detecting a first signal exceeding a first threshold, wherein thefirst signal is caused by the particles flowing through a first lobe ofthe plurality of lobes of the light pattern; detecting a second signalexceeding a second threshold, wherein the second signal is caused by theparticles flowing through a second lobe of the plurality of lobes of thelight pattern, and wherein the second threshold is greater than thefirst threshold; and based on detecting the second signal and detectingthe first signal at different times, determining one of the first andsecond signals were created by a particle of the second particles. 9.The system of claim 8, wherein the detection system is furtherconfigured for determining whether the second signal was received at apredetermined time after the first signal according to particle travelspeed between adjacent lobes of the plurality of lobes.
 10. The systemof claim 8, wherein the second particles are up to 1000 times largerthan the first particles.
 11. The system of claim 8, wherein thedetection system is configured to set the first threshold based on anenergy emission level of a particle of the first particles.
 12. Thesystem of claim 11, wherein the detection system is configured to setthe second threshold based on an energy emission level of a particle ofthe second particles.
 13. The system of claim 8, wherein the detectionsystem comprises an analog logic system.
 14. The system of claim 13,wherein the analog logic system comprise plurality of comparators.